Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil - PubMed (original) (raw)

Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil

Luca Guardabassi et al. Antimicrob Agents Chemother. 2005 Oct.

Abstract

The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.

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Figures

FIG. 1.

FIG. 1.

Schematic representation of the van operons in P. thiaminolyticus PT-2B1 (top) and P. apiarius PA-B2B (bottom) and of the PCR primers used for their characterization. The percentages refer to the levels of identity between the corresponding genes in the two operons. Open arrows represent coding sequences and indicate the direction of transcription. Solid and dashed small arrows indicate the primers used for TAIL and long PCR, respectively.

FIG. 2.

FIG. 2.

Phenotypic characteristics of P. thiaminolyticus PT-2B1. A. Atypical inhibition zone centered on a 30-μg vancomycin disk. B. Inhibition of E. faecium BM4105 by a colony of PT-2B1.

FIG. 3.

FIG. 3.

Organization of vanA operons (E. faecium B4147, accession no. M97297), vanB (E. faecalis V583, accession no. U35369), vanF (P. popilliae ATCC 14706, accession no. AF155139), _vanA_PT (P. thiaminolyticus PT-2B1, accession no. DQ018710), and vanA_PA (P. apiarius PA-B2B, accession no. DQ018711). For every gene, identity to the corresponding gene of the vanA operon in Tn_1546 and the GC content are indicated above and below the gene, respectively. Arrows indicate extent of the genes and direction of transcription.

FIG. 4.

FIG. 4.

HPLC analysis of peptidoglycan precursors of P. thiaminolyticus PT-2B1 grown without (A) or with (B) vancomycin (32 μg/ml). Samples (one-fifth of the extracts) were applied to a μ-Bondapak C18 column (300 × 3.9 mm), and isocratic elution was performed with 0.05 M ammonium phosphate (pH 4.4) at a flow rate of 0.5 ml/min. The main peaks detected by absorbance at 254 nm were identified as UDP-MurNAc-pentapeptide (1), UDP-MurNAc-pentadepsipeptide (2), and UDP-MurNAc-tetrapeptide (3) and quantitated by their uridine content. Peak 1 in panel A, 1.2 nmol; peak 2 in panel A, 0.2 nmol; peak 2 in panel B, 1.5 nmol; peak 3 in panel B, 0.9 nmol.

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